The 2016 outbreak of Zika virus (ZIKV) in the Americas demonstrated how quickly and dramatically a mosquito-borne viral infection can affect human life. Like related flavivirus family members such as West Nile virus, ZIKV can invade and infect the central nervous system (CNS), but is unique in causing in utero infection which leads to developmental abnormalities including microcephaly. ZIKV seems to have a predilection for infecting neural progenitor cells (NPCs) and persists for months in the fetal CNS after in utero infection, which suggests an ineffective immune control of the virus. We hypothesize that impaired innate immunity in neural progenitor cells underlies increased susceptibility to infection by ZIKV and contributes to microcephaly. The innate immune system includes pattern recognition receptors (PRRs) that detect pathogens and signal through effector molecules including interferon (IFN), which drives the expression of hundreds of antiviral interferon-stimulated genes (ISGs). Using induced neural progenitor cells (iNPCs) as a model for fetal CNS development, we have identified key innate immune signals that are attenuated in neural progenitors compared to mature neurons and glia: the expression levels of retinoic acid-inducible gene I (RIG-I, a PRR that detects viral RNA); and the IFN-driven upregulation of two ISGs (IFIT1 and IFITM1). We now propose to extend these findings as follows: (1) we will define the developmental changes in expression and function of PRRs during neural differentiation using the iNPC system and an established embryonic stem cell line as a control; (2) we will use CRISPR knockout or overexpression of RIG-I in bulk iNPCs and in a cerebral organoid model to test whether insufficient RIG-I signaling underlies iNPC susceptibility to ZIKV infection and microcephaly; (3) we will perform single cell RNA-seq on ZIKV-infected neural progenitors, neurons and glia to identify differentially expressed genes and gene networks, revealing innate immune components that confer susceptibility or resistance to ZIKV; and (4) using CRISPR knockout or overexpression of IFIT1 and IFITM1 we will test the role of these proteins in limiting ZIKV infection in progenitors and cerebral organoids. These experiments will define key innate immune proteins that influence susceptibility or resistance to ZIKV, identifying therapeutic targets to protect the fetal brain during ZIKV infection. Dr. Stokes? development plan builds on a background in neurosciences with coursework and hands-on training in immunology, neural stem cells, and bioinformatics. The proposal establishes a mentoring committee including faculty in immunology, neurosciences, and infectious disease to provide guidance and career development. A K08 award will allow Dr. Stokes to make maximal use of UW?s extensive scientific resources to achieve scientific independence, advancing his career goal to develop therapeutic interventions that protect neural function from injury during viral encephalitis.